U.S. patent application number 10/811140 was filed with the patent office on 2005-05-12 for in vitro model for gastrointestinal inflammation.
This patent application is currently assigned to Enterprise Ireland (trading as BioResearch Ireland). Invention is credited to Collins, John Kevin, O'Mahony, Liam.
Application Number | 20050100971 10/811140 |
Document ID | / |
Family ID | 11042182 |
Filed Date | 2005-05-12 |
United States Patent
Application |
20050100971 |
Kind Code |
A1 |
Collins, John Kevin ; et
al. |
May 12, 2005 |
In vitro model for gastrointestinal inflammation
Abstract
An in vitro for testing the immunomodulatory especially
anti-inflammatory or pro-inflammatory effect of a test material
comprises placing a microporous support having a monolayer of
epithelial cells therein in contact with a nutrient medium in a
culture well. Cells of the immune system such as peripheral blood
mononuclear cells are introduced into the medium and a test
material is also introduced into the nutrient medium. A change in
an immunological marker, especially a cytokine, particularly
TNF&agr; or IL-8 in response to the test material is
determined. The test material may be a strain of Lactobacillus or
Bifidobacterium or other material which may be a probiotic.
Inventors: |
Collins, John Kevin; (Cork,
IE) ; O'Mahony, Liam; (Cork, IE) |
Correspondence
Address: |
JACOBSON HOLMAN PLLC
400 SEVENTH STREET N.W.
SUITE 600
WASHINGTON
DC
20004
US
|
Assignee: |
Enterprise Ireland (trading as
BioResearch Ireland)
National University of Ireland, Cork.
|
Family ID: |
11042182 |
Appl. No.: |
10/811140 |
Filed: |
March 29, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10811140 |
Mar 29, 2004 |
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10173577 |
Jun 18, 2002 |
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10173577 |
Jun 18, 2002 |
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09903690 |
Jul 13, 2001 |
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09903690 |
Jul 13, 2001 |
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PCT/IE00/00009 |
Jan 17, 2000 |
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Current U.S.
Class: |
435/7.21 ;
514/12.2 |
Current CPC
Class: |
A23Y 2220/77 20130101;
G01N 33/5047 20130101; A23C 9/1234 20130101 |
Class at
Publication: |
435/007.21 ;
514/002 |
International
Class: |
G01N 033/567; A61K
038/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 15, 1999 |
IE |
990033 |
Sep 20, 1999 |
IE |
990782 |
Claims
1-15. (canceled)
16. An in vitro method for testing the inflammatory effect of a
test material comprising introducing a test material comprising an
inflammatory agent into a system comprising epithelial cells of
gastrointestinal, respiratory or genitourinary origin which
interact with the immune system and cells of the immune system and
determining the change in an immunological marker in response to
the test material, the cells which interact with the immune system
being on a microporous support.
17. The method of claim 16, wherein the cells of the immune system
are peripheral blood mononuclear cells.
18. The method of claim 16, wherein the cells which interact with
the immune system and the immune system cells are of matched
origin.
19. The method of claim 18, wherein the cells of the immune system
are of gastrointestinal, respiratory or genitourinary origin.
20. The method of claim 16, wherein the cells which interact with
the immune system are in the form of a monolayer.
21. The method of claim 16, wherein the immunological marker is a
cytokine.
22. The method of claim 21, wherein the cytokine is TNF.alpha..
23. The method of claim 21, wherein the cytokine is IL8.
24. The method of claim 16, wherein the inflammatory effect is an
anti-inflammatory effect.
25. The method of claim 16, wherein the inflammatory effect is a
proinflammatory effect.
26. The method of claim 16, wherein the test material is a
probiotic material.
27. The method of claim 16, wherein the test material includes a
strain of Bifidobacterium.
28. The method of claim 16, wherein the test material includes a
strain of Lactobacillus.
29. A method for testing the inflammatory effect of a test material
comprising the steps of: placing a microporous support having a
layer of epithelial cells of gastrointestinal, respiratory or
genitourinary origin thereon which interact with the immune system
in contact with a nutrient medium in a culture well; introducing a
composition containing cells of the immune system to the medium;
subsequently introducing a test material comprising or suspected of
comprising an inflammatory agent to either one or both cells; and
determining the change in an immunological marker in response to
the test material.
30. The method of claim 29, wherein the cells of the immune system
are peripheral blood mononuclear cells.
31. The method of claim 29, wherein the epithelial cells and the
immune system cells are of matched origin.
32. The method of claim 31, wherein the cells of the immune system
are of gastrointestinal, respiratory or genitourinary origin.
33. The method of claim 29, wherein the layer is a monolayer.
34. The method of claim 29, wherein the immunological marker is a
cytokine.
35. The method of claim 34, wherein the cytokine is TNF.alpha..
36. The method of claim 34, wherein the cytokine is IL8.
37. The method of claim 29, wherein the inflammatory effect is an
anti-inflammatory effect.
38. The method of claim 29, wherein the inflammatory effect is a
proinflammatory effect.
39. The method of claim 29, wherein the test material is a
probiotic material.
40. The method of claim 29, wherein the test material includes a
strain of Bifidobacterium.
41. The method of claim 29, wherein the test material includes a
strain of Lactobacillus.
Description
[0001] The invention relates to a system for studying intercellular
communication and in particular to an in vitro model for screening
for promoters and inhibitors of inflammation.
[0002] The problem of providing suitable model systems to mimic in
vivo normal and disease states is well known.
[0003] An improved tissue culture apparatus, the Transwell.TM. has
been developed and is described in U.S. Pat. No. 5,026,649. In this
system cells or tissue samples are separated from a nutrient medium
by a permeable membrane. A concentration gradient of nutrients may
then develop and feed the cells through the permeable membrane more
closely reflecting the situation in vivo. The Transwell.TM. has
been used to mimic various in vivo normal and disease states.
[0004] Gastrointestinal inflammation has become one of the major
health concerns in the western world. There is therefore a need to
provide an in vitro model to mimic the in vivo inflammation state
for screening for promoters and inhibitors of inflammation.
[0005] Inflammation is the term used to describe the local
accumulation of fluid, plasma proteins and white blood cells at a
site that has sustained physical damage, infection or where there
is an ongoing immune response. Inflammation is traditionally
defined by the Latin words, dolor, rubor, calor and tumor meaning
pain, redness, heat and swelling. Dilation and increased
permeability of local blood vessels, due to vasoactive plasma
proteins, reactive oxygen intermediates, nitrous oxide, certain
products of arachidonic acid metabolism, histamine and serotonin,
allows or increased blood flow and vascular permeability leading to
the redness, heat and swelling. Endothelial adhesive properties
also change causing circulating white blood cells to stick allowing
migration into the inflammatory site. The release of mediators,
such as bradykinin, within the inflammatory site causes the pain.
The first cells to arrive at an inflammatory site include the
phagocytic neutrophils followed by monocytes and later by
lymphocytes that have been activated in lymph nodes draining the
site. During an inflammatory state certain systemic effects such as
fever and acute phase protein production can also be observed.
Following eradication of the offending organism and repair of the
damaged tissue, the inflammation normally subsides.
[0006] Control of the inflammatory response is exerted on a number
of levels. The controlling factors include hormones (e.g.
hydrocortisone), prostaglandins, reactive oxygen and nitrogen
intermediates, leukotrienes and cytokines. Cytokines are low
molecular weight biologically active proteins that are involved in
the generation and control of immunological and inflammatory
responses, while also regulating development, tissue repair and
haematopoiesis. They provide a means of communication between
leukocytes themselves and also with other cell types. A number of
cell types produce these cytokines. Neutrophils, monocytes and
lymphocytes are the major sources of these cytokines during
inflammatory reactions due to their large numbers at the injured
site. Other resident cell types may also produce cytokines. For
example, injury to the skin results in keratinocyte release of IL-1
(Kupper, T. S. J Clin Invest 1990 December;86(6):1783-9). Platelets
contain stores of TGF.beta., PDGF and EGF and damage to endothelial
cells results in platelet aggregation and release of stored
cytokine.
[0007] Multiple mechanisms exist by which cytokines generated at
inflammatory sites influence the inflammatory response. Chemotaxis
stimulates homing of inflamatory cells to the injured site. A
number of cytokines are chemotactic factors, such as TGF.beta.
(Brandes M E, et al. J Immunol 1991 Sep. 1;147(5):1600-6) and IL-8
(Baggiolini M, et al. Int J Immunopharmacol 1995
February;17(2):103-8). Factors other than cytokines are also
chemotactic, such as the complement component C5a and leukotriene
B.sub.4. Cytokines promote infiltration of cells into tissue by
increasing expression of adhesion molecules on endothelial cells
and leukocytes (Springer T A. Scand J Immunol 1990
September;32(3):211-6). Cytokines released within the injured
tissue result in activation of the inflammatory infiltrate.
Stimulation of the release of reactive oxygen intermediates,
protease containing granules and additional cytokines from
monocytes and neutrophils maintain the inflammatory state. Most
cytokines are pleiotrophic and express multiple biologically
overlapping activities. Cytokine cascades and networks control the
inflammatory response rather than the action of a particular
cytokine on a particular cell type (Arai K I, et al. Annu Rev
Biochem 1990;59:783-836). However, TNF.alpha. and IL-1.beta. have
been demonstrated to be the pivotal cytokine mediators in
stimulating inflammatory responses. Waning of the inflammatory
response results in lower concentrations of the appropriate
activating signals and other inflammatory mediators leading to the
cessation of the inflammatory response. However, the factors that
control the resolution of inflammatory lesions are very poorly
understood.
[0008] The inflammatory response is critical to the defence and
repair of host tissues. However, uncontrolled responses can result
in significant tissue and organ damage and may result in the death
of the host (Dinarello C A, et al., Rev Infect Dis 1988
January-February;10(1):168-89- ). Examples of such disease states
include inflammatory bowel disease, rheumatoid arthritis, sepsis,
psoriasis, contact dermatitis, multiple sclerosis, atherosclerosis,
sarcoidosis, idiopathic pulmonary fibrosis, dermatomyositis,
hepatitis, diabetes, allograft rejection, graft versus host disease
and certain para-neoplastic phenomena. Thus, inhibition of these
destructive inflammatory responses is a compelling therapeutic
option.
[0009] As many cytokines may have both pro and anti-inflammatory
activities, patterns or networks of cytokine release have been
associated with different types of immune responses. The existence
of T cells which differ in their pattern of cytokine secretion
allows differentiation of inflammatory or immune responses into at
least two categories, cell mediated or humoral responses (for
review see Abbas A K, et al., Nature 1996 Oct.
31;383(6603):787-93). Th1 responses are categorised by IFN.gamma.,
TNF.beta. and IL-2 production leading to a cell-mediated response
while Th2 cells secrete IL-4, IL-5, IL-9, IL-10 and IL-13 resulting
in a humoral response (Mosmann T R, et al., J Immunol 1986 Apr.
1;136(7):2348-57). Differentiation of T cells into either network
depends on the cytokine milieu in which the original antigen
priming occurs (Seder R A, et al., J Exp Med 1992 Oct.
1;176(4):1091-8). These primary immune responses may be influenced
by a number of cell types including .gamma..delta. T cells (Ferrick
D A, et al., Nature 1995 Jan. 19;373(6511):255-7) and also mast
cells, which at mucosal sites seem to contain IL-4, IL-10 and IL-13
which are anti-inflammatory and Th2 type cytokines (Marietta E V,
et al., Eur J Immunol 1996 January;26(1):49-56). Different types of
stimulation may also direct this response such as immune complex
deposition within inflammatory sites, which increase IL-6 and IL-10
production and inhibits production of TNF.alpha. and IL-1.beta.
thus influencing the Th1/Th2 balance (Berger S; et al., Eur J
Immunol 1996 June;26(6):1297-301.). For successful elimination of
some pathogens, the correct cytokine network needs to be
established, such as the intracellular bacterium Listeria
monocytogenes which elicits a Th1 response while the extracellular
parasite Nippostrongylus brasiliensis requires a Th2 response
(derrick et al., 1995). Each of these T cell subsets produce
cytokines that are autocrine growth factors for that subset and
promote differentiation of naive T cells into that subset (for
review see Trinchieri G, et al., Cytokine Growth Factor Rev 1996
August; 7(2):123-32). These two subsets also produce cytokines that
cross-regulate each other's development and activity. IFN.gamma.
amplifies Th1 development and inhibits proliferation of Th2 T cells
(Fitch F W, et al., Annu Rev Immunol 1993;11:29-48) while IL-10
blocks Th1 activation.
[0010] The production of these multifunctional cytokines across a
wide spectrum of tumour types suggests that significant
inflammatory responses are ongoing in patients with cancer. It is
currently unclear what protective effect this response has against
the growth and development of tumour cells in vivo. However, these
inflammatory responses could adversely affect the tumour bearing
host. Complex cytokine interactions are involved in the regulation
of cytokine production and cell proliferation within tumour and
normal tissues (McGee D W, et al., Immunology 1995
September;86(1):6-11, 1995, Wu S, et al., Gynecol Oncol 1994
April;53(1):59-63). It has long been recognised that weight loss
(cachexia) is the single most common cause of death in patients
with cancer (Inagaki J, et al., Cancer 1974 February;33(2):568-73.)
and initial malnutrition indicates a poor prognosis (Van Eys J.
Nutr Rev 1982 December;40(12):353-9). For a tumour to grow and
spread it must induce the formation of new blood vessels and
degrade the extracellular matrix. The inflammatory response may
have significant roles to play in the above mechanisms, thus
contributing to the decline of the host and progression of the
tumour.
[0011] Thus, the initiation of inflammatory responses, and the
subsequent tissue damage, is dependent on environmental factors
such as the inflammatory agent itself, the specific cell types
present and the cytokine milieu initially present. In vitro systems
must therefore incorporate all these conditions in order to
reliably assess the inflammatory potential of novel compounds.
Currently, these in vitro systems use isolated populations of
immune cells, such as peripheral blood mononuclear cells
(PBMCs).
[0012] The present invention is directed to a cell culture system
to serve as a model for gastrointestinal inflammation and a method
for investigating intercellular communication and for screening for
promoters and inhibitors of inflammation.
STATEMENTS OF INVENTION
[0013] According to the invention there is provided a method for
testing the effect of a test material comprising introducing a test
material comprising or suspected of comprising an immunomodulatory
agent into a system comprising cells which interact with the immune
system and cells of the immune system and determining the change in
an immunological marker in response to the test material.
[0014] Preferably the cells which interact with the immune system
and the immune system cells are of matched origin.
[0015] In one embodiment of the invention the cells which interact
with the immune system are of gastrointestinal, respiratory or
genitourinary origin.
[0016] In another embodiment the cells of the immune system are of
gastrointestinal, respiratory or genitourinary origin.
[0017] Preferably the cells which interact with the immune system
are in the form of a monolayer. Most preferably the cells are on a
microporous support.
[0018] In one embodiment the invention provides a method for
testing the immunomodulatory effect of a test material comprising
the steps of:--
[0019] placing a microporous support having a layer of cells
thereon which interact with the immune system in contact with a
nutrient medium in a culture well;
[0020] introducing a composition containing cells of the immune
system to the medium;
[0021] subsequently introducing a test material comprising or
suspected of comprising an immunomodulatory agent to either one or
both cells; and
[0022] determining the change in an immunological marker in
response to the test material.
[0023] Preferably the cells which interact with immune system are
epithelial cells and the cells of the immune system are preferably
peripheral blood mononuclear cells.
[0024] Ideally the immunological marker is a cytokine such as
TNF.alpha. or IL8.
[0025] In one embodiment the invention provides a method wherein
the immunomodulatory effect is an anti-inflammatory effect.
[0026] In another embodiment the invention provides a method
wherein the immunomodulatory effect is a pro-inflammatory
effect.
[0027] Preferably the invention provides a method wherein the test
material is a material that is or suspected to be a probiotic
material.
[0028] Most preferably the test material includes a strain of
Bifidobacterium. Such strains are referred to in a concurrently
filed PCT Application filed on January 17 with the present
application.
[0029] One strain of Bifidobacterium is Bifidobacterium longum
infantis UCC 35624 or a mutant or variant thereof. A deposit of
Bifidobacterium longum infantis strain UCC 35624 was made at The
National Collections of Industrial and Marine Bacteria Limited
(NCIMB) on Jan. 13, 1999 and accorded the accession number NCIMB
41003.
[0030] Preferably the test material includes a strain of
lactobacillus. Strains of Lactobacillus salivarius are disclosed in
WO 98/35014.
[0031] One strain of Lactobacillus salvarius is Lactobacillus
salivarius strain UCC 118 or a mutant or variant thereof. A deposit
of Lactobacillus salivarius strain UCC 118 was made at the NCIMB 43
on Nov. 27, 1996 and accorded the accession number NCIMB 40829.
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] The invention will be more clearly understood from the
following description given by way of example only with reference
to the accompanying drawings in which:--
[0033] FIG. 1 is a schematic representation of a culture system
according to the invention;
[0034] FIG. 2 is a schematic representation of a cell signalling in
the test system of the invention;
[0035] FIG. 3 is a bar chart of TNF.alpha. levels in various
culture cell systems;
[0036] FIG. 4 is a bar chart of TNF.alpha. levels in the presence
of various bacterial strains;
[0037] FIG. 5 is a bar chart of TNF.alpha. levels in patient and
control samples in the presence of peripheral blood mononuclear
cells (PBMCs) and Bifidobacterium longum infantis UCC 35624;
[0038] FIG. 6 is a bar char of TNF.alpha. and IL-8 levels in the
presence of PBMCs, Bifidobacterium longum infantis UCC 35624 and
epithelial cells; and
[0039] FIG. 7 is a graph of extracellular TNF.alpha., IL-1RA, IL-6,
sIL-6R, and IFN.alpha. levels following exposure to Lactobacillus
salivarius UCC118.
DETAILED DESCRIPTION
[0040] We have developed an in vitro cell culture model of
gastrointestinal inflammation that provides a valuable means of
examining the in vivo cellular activity during inflammation. The
system includes gastrointestinal epithelial cells as these cells
release cytokines that may have powerful influences on local immune
response. The model has been used to investigate intercellular
communication and to screen for inflammatory agents. The model has
also been used to test the role of probiotic bacteria in the
modulation of inflammatory cytokines.
[0041] The culture model of the present invention may also be
utilised for the identification of novel inhibitors and promoters
of cytokine production. Compounds have been shown by this model to
inhibit or promote the inflammatory response and thus have
therapeutic value. Detecting their ability to modulate cytokine
production identifies inhibitors or promoters. The assay of this
invention also has diagnostic utilities for detecting promoters of
inflammatory responses from within inflammatory lesions.
[0042] Referring to FIG. 1 there is illustrated a cell culture
system 1 according to the invention. A single culture well 2 of a
tissue culture cluster plate, typically having 6, 12 or 24 wells,
is shown. Such a system is commercially available from Costar and
is described in U.S. Pat. No. 5,139,951 and U.S. Pat. No.
5,272,083. The well 2 comprises side walls 3 and a base 4.
Detachably suspended in the well 2 is a support 5 having as a base
a microporous filter 6. The support 5 hangs by a flange 7 at its
upper end from the top of the well 2. The flange 7 of the support 5
positions the microporous filter 6 substantially centrally in the
well 2. Openings in the side wall of the support 5 provide access
for a pipette to add and withdraw fluid from the well.
[0043] The filter 6 when in position in the cell culture well 2,
divides the well 2 into two compartments, an upper 8 and a lower
compartment 9. The microporous filter 6 with a pore size of about 3
microns is preferably of polycarbonate but other polymers that
support epithelial cell attachment may be used. The filter acts as
a dividing barrier that permits the passage of soluble mediators
between the two compartments but does not allow cell-cell
contact.
[0044] In use, a confluent epithelial cell monolayer 10 is cultured
on the upper surface of the microporous filter 6 for 2-8 weeks
under standard growth conditions. Epithelial cells suitable for use
can be obtained from any in vivo source, including but not limited
to epithelial cells derived from the human gastrointestinal tract.
CaCo-2 cells, or derivatives thereof, are most preferred, and are
available commercially from culture collection banks such as the
ATCC and ECACC. Procedures for harvesting and culturing epithelial
cells are know in the art.
[0045] A composition containing cells of the immune system is added
to media in the culture well 2 which is in contact with the lower
surface of the microporous filter. Any suitable cellular population
can be added to the lower compartment 9, including but not limited
to PBMCs.
[0046] Following shortly after the addition of PBMCs, a composition
comprising or suspected of comprising an immunomodulatory agent is
added to the culture well. These "test molecules" can be bacterial
cells, but are not limited to bacterial components. Whole bacteria
and components thereof can be utilised. On addition of the test
material a tricellular signalling network is established. This
network is schematically illustrated in FIG. 2.
[0047] The cell culture system and signalling network described
above is used in the following examples.
EXAMPLE 1
Intercellular Signalling Assay
[0048] Epithelial CaCo-2 cells are maintained in 25 cm.sup.2
culture flasks and upon reaching 80% confluence, the cells are
enzymatically (e.g. trypsin) detached from the culture flask and
washed in serum free medium. The cells are counted using a
haemacytometer, reconstituted in culture medium and added into the
cell culture support. The cells attach themselves to the
microporous filter and grow outwards forming a monolayer.
Epithelial cell monolayers that have reached confluence for a
period of time are preferred. These cells are refed every three
days.
[0049] Peripheral blood mononuclear cells (PBMCs) harvested from
peripheral blood by density gradient centrifugation and resuspended
in culture medium are added to the culture well. The epithelial
cells and PBMCs are separated from each other by the microporous
filter which allowed the passage of soluble mediators between the
two compartments but did not allow cell-cell contact.
[0050] Following shortly after the addition of PBMCs, a composition
comprising bacteria which have been previously cultured in broth
and washed in antibiotic containing medium is added to the same
compartment. The bacteria, PBMCs and epithelial cells are
co-incubated for 72 hours at 37.degree. C. in a 5% CO.sub.2
atmosphere. Supernatants are then collected and assayed for
cytokine concentration using commercially available ELISAs.
[0051] Due to its role as a proinflammatory cytokine, TNF.alpha.
levels were measured using a Boehringer Mannheim ELISA kit
(Catologue no. 1425943). Co-incubation of PBMCs alone with bacteria
resulted in the stimulation of TNF.alpha. production. Co-incubation
of PBMCs, epithelial cells and bacteria resulted in a significant
suppression of TNF.alpha. production (FIG. 3). The results with the
tricellular assay are in stark contrast to assays with only two
cell types present. It is apparent that the presence of epithelial
cells has an immunomodulatory effect on PBMC signalling.
EXAMPLE 2
Test for Anti-Inflammatory Bacterial Strains
[0052] A number of lactic acid bacteria, which have been isolated
from the human gastrointestinal tract, were examined in this novel
assay system as inhibitors of inflammatory responses. All bacterial
strains were taken from -20.degree. C. glycerol stocks and
incubated anaerobically overnight in MRS broth and washed in
antibiotic containing medium. Epithelial cell monolayers were grown
for 6 weeks prior to the addition of PBMCs and bacterial cells.
[0053] The results of these stimulations can be observed in FIG. 4.
Relative to control cultures, two bacterial strains suppressed
TNF.alpha. production. The two strains Lactobacillus salivarius
strain UCC118, which suppressed production of TNF.alpha., is the
subject of WO-A-9835014 and a PCT Application filed concurrently
with the present application. The Bifidobacterium longum infantis
strain UCC 35624 is the subject of a PCT Application filed
concurrently with the present application.
EXAMPLE 3
In Vitro Studies to Examine the Immune Perception of
Bifidobacterium longum infantis
[0054] Overnight washed cultures of Bifidobacterium longum infantis
UCC 35624 were incubated with human peripheral blood mononuclear
cells (PBMCs) from both healthy volunteers (n=9) and patients
suffering from inflammatory bowel disease (n=5). Production of the
proinflammatory cytokine tumour necrosis factor .alpha.
(TNF.alpha.) was measured by ELISA in seventy two hour culture
supernatants. Co-incubation of Bifidobacterium longum infantis UCC
35624 with human PBMCs did not result in the stimulation of
TNF.alpha. production (FIG. 5). Thus, exposure of the systemic
immune system to this bacterium does not induce an inflammatory
response.
[0055] In order to assess the immune perception of Bifidobacterium
longum infantis UCC 35624 at mucosal surfaces, co-culturing of
epithelial cells and PBMCs was performed in transwell chambers. An
epithelial cell monolayer was grown in the upper chamber and PBMCs
were incubated in the lower compartment. These were separated from
each other by a porous membrane which allowed the passage of
soluble mediators between the two compartments but did not allow
cell-cell contact. Using this model, the production of TNF.alpha.
and Interleukin-8 (IL-8) was measured in the presence and absence
of Bifidobacterium longum infantis UCC 35624 in the PBMC
compartment. Co-culture of epithelial cells, PBMCs and
Bifidobacterium longum infantis resulted in significant suppression
of TNF.alpha. and IL-8 production (FIG. 6). Thus, a tri-cellular
network involving epithelial cells, PBMCs and Bifidobacterium
longum infantis UCC 35624 results in suppression of proinflammatory
cytokine production.
EXAMPLE 4
In Vitro Demonstration of the Mechanisms Underlying the
Anti-Inflammatory Effects of Lactobacillus salivarius Especially
Subspecies Salivarius UCC118
[0056] Using the cell culture assay system as described in examples
1 and 2, with epithelial cells as described above and peripheral
blood mononuclear cells in the one compartment, extracellular
cytokine levels were measured by ELISAs. Following co-incubation
with Lactobacillus salivarius UCC118, the amount of TNF.alpha.
produced was significantly reduced compared to control cultures.
Furthermore, IL-1RA and IFN.gamma. levels dropped while IL-6 and
soluble IL-6 receptor levels increased (FIG. 7). Intracellular
staining for TNF.alpha. confirmed the ELISA result as TNF.alpha.
levels were lower in the UCC118 stimulated sample compared to
controls.
[0057] In vitro models have demonstrated that Lactobacillus
salivarius UCC118 is capable of inducing Th2 type cytokines (i.e.
interleukin 6 and interleukin 6 soluble receptor) while suppressing
the production of inflammatory cytokines such as tumour necrosis
factor .alpha. and interleukin 1.beta.. Thus, these results suggest
that consumption of Lactobacillus salivarius UCC118 would be of
benefit to patients suffering from inflammatory diseases, such as
IBD.
[0058] It will be appreciated that while the specific test models
referred to above are used for testing the immunomodulatory
especially pro or anti-inflammatory effects of specific probiotic
materials the model may also be applied to test any probiotic
material. Indeed, the method may equally have application to any
other suitable test material such as bacterial components,
synthetic chemicals such as drugs or drug candidates, biological
compounds such as cells, components thereof, or cytokines including
IL-12, IL-10 and/or TGF-.beta..
[0059] It will also be appreciated that while the invention has
been described with reference to its application to a specific
assay system it may be applied using any suitable assay system.
[0060] The invention is not limited to the embodiments hereinbefore
described which may be varied in detail.
* * * * *